The present invention generally relates to immunotherapeutic agents an d vaccines against intracellular pathogenic organisms such as bacteria, protozoa, viruses and fingi. More specifically, unlike prior art vaccines and immunotherapeutic agents based upon pathogenic subunits, killed pathogens and attenuated natural pathogens, the present invention uses recombinant attenuated pathogens, or closely related species, that express and secrete immunogenic determinants of a selected pathogen stimulating an effective immune response in mammalian hosts. The immunostimulatory vaccines and immunotherapeutics of the present invention are derived from recombinant attenuated intracellular pathogens, or closely related species, that express immunogenic determinants in situ.
It has long been recognized that parasitic microorganisms possess the ability to infect animals thereby causing disease and often the death of the host. Pathogenic agents have been a leading cause of death throughout history and continue to inflict immense suffering. Though the last hundred years have seen dramatic advances in the prevention and treatment of many infectious diseases, complicated host-parasite interactions still limit the universal effectiveness of therapeutic measures. Difficulties in countering the sophisticated invasive mechanisms displayed by many pathogenic organisms is evidenced by the resurgence of various diseases such as tuberculosis, as well as the appearance of numerous drug resistant strains of bacteria and viruses.
Among those pathogenic agents of major epidemiological concern, intracellular bacteria have proven to be particularly intractable in the face of therapeutic or prophylactic measures. Intracellular bacteria, including the genus mycobacterium and the genus Legionella, complete all or part of their lifecycle within the cells of the infected host organism rather than extracellularly. Around the world, intracellular bacteria are responsible for millions of deaths each year and untold suffering. Tuberculosis is the leading cause of death from a single disease agent worldwide, with 10 million new cases and 2.9 million deaths every year. In addition, intracellular bacteria are responsible for millions of cases of leprosy. Other debilitating diseases transmitted by intracellular agents include cutaneous and visceral leishmaniasis, American trypanosomiasis (Chagas disease), listeriosis, toxoplasmosis, histoplasmosis, trachoma, psittacosis, Q-fever, and legionellosis. At this time, relatively little can be done to prevent debilitating infections in susceptible individuals exposed to many of these organisms Due to this inability to effectively protect populations from such intracellular pathogens and the resulting human and animal morbidity and mortality caused by such agents, tuberculosis, is one of the most important diseases now confronting mankind.
Those skilled in the art will appreciate that the following exemplary discussion of M. tuberculosis is illustrative of the teachings of the present invention and is in no way intended to limit the scope of the present invention to the treatment of M. tuberculosis. Similarly, the teachings herein are not limited in any way to the treatment of tubercular infections. On the contrary, this invention may be used to advantageously provide safe and effective vaccines and immunotherapeutic agents against any pathogenic agent by using recombinant attenuated pathogens, or recombinant avirulent organisms, to express, and of equal importance to release the immunologically important proteins of the pathogenic organism.
Currently it is believed that approximately one-third of the world""s population is infected by M. tuberculosis resulting in millions of cases of pulmonary tuberculosis annually. More specifically, human pulmonary tuberculosis primarily caused by M. tuberculosis is a major cause of death in developing countries. Capable of surviving inside macrophages and monocytes, M. tuberculosis may produce a chronic intracellular infection. M. tuberculosis is relatively successful in evading the normal defenses of the host organism by concealing itself within the cells primarily responsible for the detection of foreign elements and subsequent activation of the immune system. Moreover, many of the front-line chemotherapeutic agents used to treat tuberculosis have relatively low activity against intracellular organisms as compared to extracellular forms. These same pathogenic characteristics have heretofore prevented the development of fully effective immunotherapeutic agents or vaccines against tubercular infections.
While this disease is a particularly acute health problem in the developing countries of Latin America, Africa, and Asia, it is also becoming more prevalent in the first world. In the United States specific populations are at increased risk, especially urban poor, immunocompromised individuals and immigrants from areas of high disease prevalence. Largely due to the AIDS epidemic, in recent years the incidence of tuberculosis has increased in developed countries, often in the form of multi-drug resistant M. tuberculosis. 
Recently, tuberculosis resistance to one or more drugs was reported in 36 of the 50 United States. In New York City, one-third of all cases tested was resistant to one or more major drugs. Though non-resistant tuberculosis can be cured with a long course of antibiotics, the outlook regarding drug resistant strains is bleak. Patients infected with strains resistant to two or more major antibiotics have a fatality rate of around 50%. Accordingly, safe and effective vaccines against such varieties of M. tuberculosis are sorely needed.
Initial infections of M. tuberculosis almost always occur through the inhalation of aerosolized particles as the pathogen can remain viable for weeks or months in moist or dry sputum. Although the primary site of the infection is in the lungs, the organism can also cause infection of nearly any organ including, but not limited to, the bones, spleen, kidney, meninges and skin. Depending on the virulence of the particular strain and the resistance of the host, the infection and corresponding damage to the tissue may be minor or extensive. In the case of humans, the initial infection is controlled in the majority of individuals exposed to virulent strains of the bacteria. The development of acquired immunity following the initial challenge reduces bacterial proliferation thereby allowing lesions to heal and leaving the subject largely asymptomatic.
When M. tuberculosis is not controlled by the infected subject it often results in the extensive degradation of lung tissue. In susceptible individuals lesions are usually formed in the lung as the tubercle bacilli reproduce within alveolar or pulmonary macrophages. As the organisms multiply, they may spread through the lymphatic system to distal lymph nodes and through the blood stream to the lung apices, bone marrow, kidney and meninges surrounding the brain. Primarily as the result of cell-mediated hypersensitivity responses, characteristic granulomatous lesions or tubercles are produced in proportion to the severity of the infection. These lesions consist of epithelioid cells bordered by monocytes, lymphocytes and fibroblasts. In most instances a lesion or tubercle eventually becomes necrotic and undergoes caseation (conversion of affected tissues into a soft cheesy substance).
While M. tuberculosis is a significant pathogen, other species of the genus Mycobacterium also cause disease in animals including man and are clearly within the scope of the present invention. For example, M. bovis is closely related to M. tuberculosis and is responsible for tubercular infections in domestic animals such as cattle, pigs, sheep, horses, dogs and cats. Further, M. bovis may infect humans via the intestinal tract, typically from the ingestion of raw milk. The localized intestinal infection eventually spreads to the respiratory tract and is followed shortly by the classic symptoms of tuberculosis. Another important pathogenic vector of the genus Mycobacterium is M. leprae that causes millions of cases of the ancient disease leprosy. Other species of this genus which cause disease in animals and man include M. kansasii, M. aviuim intracellulare, M. fortuitum, M. marinum, M. chelonei, and M. scrofulaceum. The pathogenic mycobacterial species frequently exhibit a high degree of homology in their respective DNA and corresponding protein sequences and some species, such as M. tuberculosis and M. bovis, are highly related.
For obvious practical and moral reasons, initial work in humans to determine the efficacy of experimental compositions with regard to such afflictions is infeasible. Accordingly, in the early development of any drug or vaccine it is standard procedure to employ appropriate animal models for reasons of safety and expense. The success of implementing laboratory animal models is predicated on the understanding that immunogenic epitopes are frequently active in different host species. Thus, an immunogenic determinant in one species, for example a rodent or guinea pig, will generally be immunoreactive in a different species such as in humans. Only after the appropriate animal models are sufficiently developed will clinical trials in humans be carried out to further demonstrate the safety and efficacy of a vaccine in man.
With regard to alveolar or pulmonary infections by M. tuberculosis, the guinea pig model closely resembles the human pathology of the disease in many respects. Accordingly, it is well understood by those skilled in the art that it is appropriate to extrapolate the guinea pig model of this disease to humans and other mammals. As with humans, guinea pigs are susceptible to tubercular infection with low doses of the aerosolized human pathogen M. tuberculosis. Unlike humans where the initial infection is usually controlled, guinea pigs consistently develop disseminated disease upon exposure to the aerosolized pathogen, facilitating subsequent analysis. Further, both guinea pigs and humans display cutaneous delayed-type hypersensitivity reactions characterized by the development of a dense mononuclear cell induration or rigid area at the skin test site. Finally, the characteristic tubercular lesions of humans and guinea pigs exhibit similar morphology including the presence of Langhans giant cells. As guinea pigs are more susceptible to initial infection and progression of the disease than humans, any protection conferred in experiments using this animal model provides a strong indication that the same protective immunity may be generated in man or other less susceptible mammals. Accordingly, for purposes of explanation only and not for purposes of limitation, the present invention will be primarily demonstrated in the exemplary context of guinea pigs as the mammalian host. Those skilled in the art will appreciate that the present invention may be practiced with other mammalian hosts including humans and domesticated animals.
Any animal or human infected with a pathogenic organism and, in particular, an intracellular organism, presents a difficult challenge to the host immune system. While many infectious agents may be effectively controlled by the humoral response and corresponding production of protective antibodies, these mechanisms are primarily effective only against those pathogens located in the body""s extracellular fluid. In particular, opsonizing antibodies bind to extracellular foreign agents thereby rendering them susceptible to phagocytosis and subsequent intracellular killing. Yet this is not the case for other pathogens. For example, previous studies have indicated that the humoral immune response does not appear to play a significant protective role against infections by intracellular bacteria such as M. tuberculosis. However, the present invention may generate a beneficial humoral response to the target pathogen and, as such, its effectiveness is not limited to any specific component of the stimulated immune response.
More specifically, antibody mediated defenses seemingly do not prevent the initial infection of intracellular pathogens and are ineffectual once the bacteria are sequestered within the cells of the host. As water soluble proteins, antibodies can permeate the extracellular fluid and blood, but have difficulty migrating across the lipid membranes of cells. Further, the production of opsonizing antibodies against bacterial surface structures may actually assist intracellular pathogens in entering the host cell. Accordingly, any effective prophylactic measure against intracellular agents, such as Mycobacterium, should incorporate an aggressive cell-mediated immune response component leading to the rapid proliferation of antigen specific lymphocytes that activate the compromised phagocytes or cytotoxically eliminate them. However, as will be discussed in detail below, inducing a cell-mediated immune response does not equal the induction of protective immunity. Though cell-mediated immunity may be a prerequisite to protective immunity, the production of vaccines in accordance with the teachings of the present invention requires animal based challenge studies.
This cell-mediated immune response generally involves two steps. The initial step, signaling that the cell is infected, is accomplished by special molecules (major histocompatibility or MHC molecules) which deliver pieces of the pathogen to the surface of the cell. These MHC molecules bind to small fragments of bacterial proteins that have been degraded within the infected cell and present them at the surface of the cell. Their presentation to T-cells stimulates the immune system of the host to eliminate the infected host cell or induces the host cell to eradicate any bacteria residing within.
Attempts to eradicate tuberculosis using vaccination was initiated in 1921 after Calmette and Guxc3xa9rin successfully attenuated a virulent strain of M. bovis using in vitro serial passage techniques. The resultant live vaccine developed at the Institut Pasteur in Lille, France is known as the Bacille Calmette and Guxc3xa9rin, or BCG vaccine. Nearly eighty years later this vaccine remains the only prophylactic therapy for tuberculosis currently in use. In fact all BCG vaccines available today are derived from the original strain of M. bovis developed by Calmette and Guxc3xa9rin at the Institut Pasteur.
The World Health Organization considers the BCG vaccine an essential factor in reducing tuberculosis worldwide, especially in developing nations. In theory, BCG vaccine confers cell-mediated immunity against an attenuated mycobacterium that is immunologically related to M. tuberculosis. The resulting immune response should prevent primary tuberculosis. Thus, if primary tuberculosis is prevented, latent infections cannot occur and disease reactivation is avoided.
However, controlled clinical trials have revealed significant variations in vaccine efficacy. Reported efficacy rates have varied between 0-80%. Vaccine trials conducted in English school children reported a ten-year post vaccination protection rate in excess of 78%. However, in a similar trial in South India, BCG failed to protect against culture-proven primary tuberculosis in the first 5 years post inoculation. A recent meta-analysis of BCG efficacy in the prevention of tuberculosis estimated that overall prophylactic efficacy was approximately 50%. (Colditz, G. A. T. F. Brewer, C. S. Berkey, M. E. Wilson, E. Burdick, H. V. Fineberg, and F. Mosteller. 1994. JAMA 271:698-702.)
This remarkable disparity in reported efficacy rates remains a vexing problem for health officials and practitioners that must determine when and how to use the BCG vaccine. Numerous factors have been implicated that may account for these observed efficacy disparities including differences in manufacturing techniques, routes of inoculation and characteristics of the populations and environments in which the vaccines have been used. Recent work suggests that incidental contact with environmental mycobacteria may result in a xe2x80x9cnatural vaccinexe2x80x9d that prevents the vaccine recipient from mounting an effective response to native BCG proteins.
In order to minimize BCG immunogenicity variation, vaccine manufactures maintain master stocks of original vaccine strains in the lyophilized (freeze dried) state. Each production strain derived therefrom is in turn named after the manufacturing site, company or bacterial strain, for example: BCG-London, BCG-Copenhagen, BCG-Connaught, or BCG-Tice (marketed worldwide by Organon, Inc.). In an effort to standardize manufacturing techniques in the United States, the Federal Food and Drug Administration""s (FDA) Center for Biologic Education and Research (CBER) regulates vaccine manufacturing. The FDA""s CBER branch has specified that each lyophilized BCG strain used for vaccination must be capable of inducing a specified tuberculin skin test reaction in guinea pigs and humans. Unfortunately, induced tuberculin sensitivity has not been shown to correlate with protective immunity.
Current BCG vaccines are provided as lyphophilzed cultures that are re-hydrated with sterile diluent immediately before administration. The BCG vaccine is given at birth, in infancy, or in early childhood in countries that practice BCG vaccination, including developing and developed countries. Adult visitors to endemic regions who may have been exposed to high doses of infectious mycobacteria may receive BCG as a prophylactic providing they are skin test non-reactive. Adverse reactions to the vaccine are rare and are generally limited to skin ulcerations and lymphadenitis near the injection site. However, in spite of these rare adverse reactions, the BCG vaccine has an unparalleled history of safety with over three billion doses having been administered worldwide since 1930.
Eighty-years have now passed since BCG was developed and there remains paucity in acceptable vaccine alternatives. Recently, the present inventors have made considerable progress in the isolation, characterization and recombinant expression of extracellular proteins secreted by intracellular pathogens. For example, the inventors"" U.S. Pat. No. 5,108,745, issued Apr. 28, 1992 and several pending U.S. Patent applications provide vaccines and methods of producing protective immunity against L. pneumophila and M. tuberculosis as well as other intracellular pathogens. These prior art vaccines are broadly based on extracellular products originally derived from proteinaceous compounds released extracellularly by the pathogenic bacteria into broth culture in vitro and released extracellularly by bacteria within infected host cells in vivo. As provided therein, these vaccines are selectively based on the identification of extracellular products or their analogs that stimulate a strong immune response against the target pathogen in a mammalian host
Vaccines prepared from selected M. tuberculosis extracellular products are currently being optimized for use as human prophylactic therapies. Protein cocktails and individual protein preparations using both recombinant as well as naturally expressed proteins are being studied. One goal of these ongoing studies is to maximize the base immune response through optimum immunogen (protein) presentation. To date over 100 different preparations have been made including 38 different protein combinations, 26 different adjuvants, 10 different protein concentrations and seven different dosing regimens. The candidate vaccine proteins have also been coupled to non-M. tuberculosis proteins including bovine serum albumin, Legionella sp. major secretory protein, and tetanus toxoid. This list is not inclusive of methods the present inventors have used to present extracellular proteins of intracellular pathogens to host animals; rather it illustrates the enormous complexity and inherent variability associated with vaccine optimization. However, in spite of these and other activities, no combination of extracellular proteins, adjuvants, carrier proteins, concentrations or dosing frequencies resulted in inducing a protective immune response in guinea pigs that was comparable or superior to BCG.
Recently, significant attention has been focused on using transformed BCG strains to produce vaccines that express various cell-associated antigens. For example, C. K. Stover, et al. have reported a Lyme Disease vaccine using a recombinant BCG (rBCG) that expresses the membrane associated lipoprotein OspA of Borrelia burgdorferi. Similarly, the same author has also produced a rBCG vaccine expressing a pneumococcal surface protein (PsPA) of Streptococcus pneumoniae. (Stover, C. K., G. P. Bansal, S. Langerman, and M. S. Hanson. 1994. Protective Immunity Elicited by rBCG Vaccines. In: Brown F. (ed): Recombinant Vectors in Vaccine Development. Dev Biol Stand. Dasel, Karger, Vol. 82, 163-170.)
U.S. Pat. No. 5,504,005 (the xe2x80x9c""005xe2x80x9d patentxe2x80x9d) and U.S. Pat. No. 5,854,055 (the xe2x80x9c""055 patentxe2x80x9d) both issued to B. R. Bloom et al., disclose theoretical rBCG vectors expressing a wide range of cell associated fusion proteins from numerous species of microorganisms. The theoretical vectors described in these patents are either directed to cell associated fusion proteins, as opposed to extracellular non-fusion protein antigens, and/or the rBCG is hypothetically expressing fusion proteins from distantly related species. Moreover, the recombinant cell associated fusion proteins expressed in these models are encoded on DNA that is integrated into the host genome and under the control of heat shock promoters. Consequently, the antigens expressed are fusion proteins and expression is limited to levels approximately equal to, or less than, the vector""s native proteins.
Furthermore, neither the ""005 nor the ""055 patent disclose animal model safety testing, immune response development or protective immunity in an animal system that closely emulates human disease. In addition, only theoretical rBCG vectors expressing M. tuberculosis fusion proteins are disclosed in the ""005 and ""055, no actual vaccines are enabled. Those vaccine models for M. tuberculosis that are disclosed are directed to cell associated heat shock fusion proteins, not extracellular non-fusion proteins.
U.S. Pat. No. 5,830,475 (the xe2x80x9c""475 patentxe2x80x9d) also discloses theoretical mycobacterial vaccines used to express fusion proteins. The DNA encoding for these fusion proteins resides in extrachromasomal plasmids under the control of mycobacterial heat shock protein and stress protein promoters. The vaccines disclosed are intended to elicit immune responses in non-human animals for the purpose of producing antibodies thereto and not shown to prevent intracellular pathogen diseases in mammals. Moreover, the ""475 patent does not disclose recombinant vaccinating agents that use protein specific promoters to express extracellular non-fusion proteins.
The present inventors propose, without limitation, that major extracellular non-fusion proteins of intracellular pathogens may be important immunoprotective molecules. First extracellular non-fusion proteins, by virtue of their release by the pathogen into the intracellular milieu of the host cell, are available for processing and presentation to the immune system as fragments bound to MHC molecules on the host cell surface. These peptide-MHC complexes serve to alert the immune system to the presence within the host cell of an otherwise hidden invader, enabling the immune system to mount an appropriate anti-microbial attack against the invader. Second, effective immunization with extracellular proteins is able to induce a population of immune cells that recognize the same peptide-MHC complexes at some future time when the complexes are displayed on host cells invaded by the relevant intracellular pathogen. The immune cells are thus able to target the infected host cells and either activate them with cytokines, thereby enabling them to restrict growth of the intracellular pathogen, or lyse them, thereby denying the pathogen the intracellular milieu in which it thrives. Third, among the extracellular proteins, the major ones, i.e., those produced most abundantly, will figure most prominently as immunoprotective molecules since they would generally provide the richest display of peptide-MHC complexes to the immune system
Therefore, there remains a need for recombinant intracellular pathogen vaccines that express major extracellular non-fusion proteins of intracellular pathogens that are closely related to the vaccinating agent. Furthermore, there is a need for recombinant intracellular pathogen vaccines that are capable of over-expressing recombinant extracellular non-fusion proteins by virtue of extrachromosomal DNA having non-heat shock gene promoters or non-stress protein gene promoters.
Specifically, there remains an urgent need to produce intracellular pathogen vaccines that provide recipients protection from diseases that is superior to the protection afforded BCG vaccine recipients. Moreover, there is an urgent need to provide both developed and developing countries with a cost efficient, immunotherapeutic and prophylactic treatment for tuberculosis and other intracellular pathogens.
Therefore, it is an object of the present invention to provide therapeutic and prophylactic vaccines for the treatment and prevention of disease caused by intracellular pathogens.
It is another object of the present invention to provide vaccines for preventing intracellular pathogen diseases using intracellular pathogens that have been transformed to express the major recombinant immunogenic antigens of the same intracellular pathogen, another intracellular pathogen, or both.
It is yet another object of the present invention to provide vaccines for the treatment and prevention of mycobacteria diseases using recombinant BCG that expresses the extracellular protein(s) of a pathogenic mycobacterium.
It is another object of the present invention to provide vaccines for treatment and/or prevention of tuberculosis using recombinant strains of BCG that express and secrete one or more major extracellular proteins of Mycobacterium tuberculosis. 
The present invention accomplishes the above-described and other objects by providing a new class of vaccines and immunotherapeutics and methods for treating and preventing intracellular pathogen diseases in mammals. Historically intracellular pathogen vaccines and immunotherapeutics have been prepared from the intracellular pathogen itself or a closely related species. These old vaccine models were composed of the entire microorganism or subunits thereof. For example, the first, and currently only available vaccine, for Mycobacterium tuberculosis is an attenuated live vaccine made from the closely related intracellular pathogen M. bovis. Recently, the present inventors have discovered that specific extracellular products of intracellular pathogens that are secreted into growth media can be used to illicit protective immune responses in mammals either as individual subunits, or in subunit combinations. However, these subunit vaccines have not proven to be superior to the original attenuated vaccine derived from M. bovis. 
The present invention details vaccines and immunotherapeutics composed of recombinant attenuated intracellular pathogens (vaccinating agents) that have been transformed to express the extracellular protein(s) (recombinant immunogenic antigens) of another or same intracellular pathogen. In one embodiment the vaccines of the present invention are made using recombinant strains of the Bacille Calmette and Guxc3xa9rin, or BCG. In this embodiment the recombinant BCG expresses major extracellular proteins of pathogenic mycobacteria including, but not limited to, M. tuberculosis, M. leprae and M. bovis, to name but a few.
The major extracellular proteins expressed by the recombinant BCG include, but are not limited to, the 12 kDa, 14 kDa, 16 kDa, 23 kDa, 23.5 kDa, 30 kDa, 32A kDa, 32B kDa, 45 kDa, 58 kDa, 71 kDa, 80 kDa, and 110 kDa of Mycobacterium sp. and respective analogs, homologs and subunits thereof including recombinant non-fusion proteins, fusion proteins and derivatives thereof. It is apparent to those of ordinary skill in the at that the molecular weights used to identify the major extracellular proteins of Mycobacteria and other intracellular pathogens are only intended to be approximations. Those skilled in the art of recombinant technology and molecular biology will realize that it is possible to co-express (co-translate) these proteins with additional amino acids, polypeptides and proteins, as it its also possible to express these proteins in truncated forms. The resulting modified proteins are still considered to be within the scope of the present invention whether termed native, non-fusion proteins, fusion proteins, hybrid proteins or chimeric proteins. For the purposes of the present invention, fusion proteins are defined to include, but not limited to, the products of two or more coding sequences from different genes that have been cloned together and that, after translation, form a single polypeptide sequence.
The present invention also describes recombinant attenuated intracellular pathogen vaccinating agents that over express non-fusion proteins from at least one other intracellular pathogen. This is accomplished by using extrachromosomal nucleic acids to express at least one recombinant immunogenic antigen gene and placing this gene(s) under the control of non-heat shock gene promoters or non-stess protein gene promoters, preferably protein-specific promoter sequences. Consequently, vaccines are provided having non-fusion, recombinant immunogenic antigens expressed in greater quantities than possible when genes encoding for recombinant immunogenic antigens are stably integrated into the vaccinating agent""s genomic DNA. As a result, intracellular pathogen vaccines having surprisingly superior specificity and potency than existing subunit or attenuated intracellular pathogen vaccines are provided.
Moreover the present invention describes methods of treating and preventing mammalian diseases caused by intracellular pathogens using the vaccines of the present invention. A partial list of the many intracellular pathogens that may be used as the attenuated vaccinating agents and/or the source of the recombinant immunogenic antigens includes, but is not limited to, Mycobacterium bovis, M. tuberculosis, M. leprae, M. kasasii, M. avium, Mycobacterium sp., Legionella pneumophila, L. longbeachae, L. bozemanii, Legionella sp., Ricketsia rickettsii, Rickettsia typhi, Rickettsia sp., Ehrlichia chaffeensis, Ehrlichia phagocytophila geno group, Ehrlichia sp., Coxiella burnetii, Leishmania sp, Toxpolasma gondii, Trypanosoma cruzi, Chlamydia pneumoniae, Chlamydia sp, Listeria monocytogenes, Listeria sp, and Histoplasma sp. In one embodiment of the present invention a recombinant BCG expressing the 30 kDa major extracellular protein of M. tuberculosis is administered to mammals using intradermal inoculations. However, it is understood that the vaccines of the present invention may be administered using any approach that will result in the appropriate immune response including, but not limited to, subcutaneous, intramuscular, intranasal, intraperitoneal, oral, or inhalation. Following a suitable post inoculation period, the mammals were challenged with an infectious M. tuberculosis aerosol. Mammals receiving the vaccine of the present invention were remarkably disease free as compared to mammals receiving BCG alone, the major extracellular protein alone, or any combinations thereof
Other objects and features and advantages of the present invention will be apparent to those skilled in the art from a consideration of the following detailed description of preferred exemplary embodiments thereof taken in conjunction with the Figures which will first be described briefly.